Special Issue:

Advancements in Animal Health and Production in Low and Middle-Income Countries

Prevalence, Biofilm Formation, Antimicrobial Resistance, and Immune Response of Staphylococcus in the Milk of Dairy Cattle

Ali Dhiaa Marza1*, Amer Jebur Obayes Al-Isawi2, Noor R. Abady3, Ahmed Hassan salh1, Faez Firdaus Jesse Abdullah4,5

1Department of Veterinary Internal Medicine, College of Veterinary Medicine, Al-Qasim Green University, Iraq; 2Department of Veterinary Public Health, College of Veterinary Medicine, Al-Qasim Green University, Iraq; 3Department of Microbiology, College of Veterinary Medicine, Al-Qasim Green University, Iraq; 4Department of Veterinary Clinical Studies, Faculty of Veterinary Medicine, Universiti Putra Malaysia, 43400, Serdang, Selangor, Malaysia; 5Research Centre for Ruminant Diseases, Faculty of Veterinary Medicine, Universiti Putra Malaysia, 43400 Serdang, Malaysia.

Received | June 27, 2025; Accepted | August 06, 2025; Published | August 16, 2025

*Correspondence | Ali Dhiaa Marza, Department of Veterinary Internal Medicine, College of Veterinary Medicine, Al-Qasim Green University, Iraq; Email: [email protected]

Citation | Marza AD, Al-Isawi AJO, Abady NR, Salh AH, Abdullah FFJ (2025). Prevalence, biofilm formation, antimicrobial resistance, and immune response of Staphylococcus in the milk of dairy cattle. J. Anim. Health Prod. 13(s1): 167-172.

DOI | https://dx.doi.org/10.17582/journal.jahp/2024/13.s1.167.172

ISSN (Online) | 2308-2801

Copyright: 2025 by the authors. Licensee ResearchersLinks Ltd, England, UK.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

Introduction

Milk is a cornerstone of human nutrition, providing essential proteins, fats, vitamins, and minerals, and serving as the basis for numerous dairy products consumed worldwide (Mekouar, 2021). However, its physicochemical properties including high water activity and neutral Ph render it highly vulnerable to microbial contamination at every stage, from production to consumption (Pajohesh et al., 2022; Di Domenico et al., 2018). This contamination not only compromises product quality and shelf life but also poses significant health risks, particularly to infants, older people, and individuals with compromised immune systems (Chung et al., 2015). Among the diverse microbial hazards associated with raw milk, Staphylococcus aureus and Staphylococcus epidermidis are particularly concerning. S. aureus is a primary causative agent of bovine mastitis, resulting in significant economic losses in dairy herds and posing a potential risk for the transmission of enterotoxins and antibiotic-resistant strains to humans through the food chain (Eidaroos et al., 2025; Niedziela et al., 2021). The ability of staphylococci to form biofilms further enhances their persistence in dairy environments, complicating sanitation efforts and facilitating the survival and dissemination of resistant clones (Singh et al., 2023; Qiu et al., 2022; Lianou et al., 2021). Recent global trends indicate a worrying rise in antimicrobial resistance among foodborne pathogens, including staphylococci isolated from livestock, which threatens the efficacy of critical antibiotics such as fluoroquinolones and aminoglycosides (Lianou et al., 2021; Pajohesh et al., 2022; Ali et al., 2024; Al-Sailawi et al., 2024).

In Iraq and similar settings, inconsistent mastitis control protocols and limited laboratory resources may exacerbate this trend, yet local data on resistance patterns and biofilm prevalence remain scarce. Beyond direct infection risks, staphylococcal contamination of milk can trigger robust host immune responses, as reflected by elevated levels of cytokines such as interleukin-6 (IL-6) and interleukin-10 (IL-10), which serve as biomarkers of inflammation and immune modulation (Bochniarz et al., 2017; Jesse et al., 2017; Wang et al., 2000). Understanding these immunological profiles is crucial for developing diagnostic and intervention strategies, particularly within the One Health framework, which recognizes the interconnectedness of animal, human, and environmental health (Abdullahi et al., 2021; Algammal et al., 2020). Despite the recognized public health importance of staphylococcal contamination in raw milk, there is a paucity of region-specific data on the prevalence, biofilm-forming capacity, antimicrobial resistance, and immunological impact of these pathogens in Iraq. Addressing this gap is essential for informing evidence-based interventions and guiding policy decisions. Therefore, this study was designed to assess the prevalence, biofilm production, antimicrobial resistance profiles, and cytokine response patterns of S. aureus and S. epidermidis isolated from raw milk in commercial dairy farms in Babylon Governorate, Iraq.

We hypothesize that raw milk in this setting serves as a reservoir for pathogenic, biofilm-forming, and antibiotic-resistant staphylococci, which are capable of eliciting significant immune responses and thus pose a multifaceted risk to dairy safety and public health. The findings are expected to inform local and regional surveillance, hygiene practices, and antimicrobial stewardship policies, aligning with the principles of One Health.

Materials and Methods

Sample collection and preparation

Forty raw milk samples were aseptically collected from commercial dairy farms in Babylon Governorate, Iraq, using sterile 50 mL conical tubes (Falcon™). Before sampling, cow teats were disinfected with 70% ethanol to minimize external contamination. Samples were immediately stored at 4°C and transported to the laboratory within 4 hours to preserve microbial integrity. Each sample was gently inverted ten times before to analysis to ensure homogeneity (Middleton et al., 2014).

Microbiological analysis

Bacterial isolation

For primary isolation, 100 μL aliquots from each milk sample were streaked onto Mannitol Salt Agar (MSA; Oxoid CM0085) and Congo Red Agar (CRA), the latter supplemented with 0.8 g/L Congo red dye (Kateete et al., 2010). Plates were incubated aerobically at 37°C for 24–48 hours.

Bacterial identification

Presumptive Staphylococcus colonies were identified using a three-step protocol. First involved an assessment of colony morphology, with mannitol-fermenting S. aureus appearing yellow on MSA and non-fermenters such as S. epidermidis as pink/red. The second step involved Gram staining using Hucker’s method to confirm Gram-positive cocci, while the third one; included biochemical testing, including catalase (3% H₂O₂) and tube coagulase (rabbit plasma) assays (Rai et al., 2023). For definitive identification, the API Staph system (bioMérieux, France) was used according to the manufacturer’s instructions.

Biofilm formation assessment

Biofilm production was first screened qualitatively on CRA plates. After 48 hours of incubation at 37°C, biofilm-producing strains were identified by the formation of black, dry, rough colonies, while non-producers formed smooth red colonies (Kaiser et al., 2013). For quantitative assessment, a microtiter plate assay was performed: overnight cultures were diluted 1:100 in tryptic soy broth supplemented with 1% glucose, inoculated into sterile 96-well polystyrene plates, and incubated at 37°C for 24 hours. Wells were washed with phosphate-buffered saline (PBS), fixed with methanol, stained with 0.1% crystal violet, and the bound dye was solubilized with 95% ethanol. Optical density (OD) was measured at 570 nm using a microplate reader (BioTek ELx808). Isolates were categorized as strong, moderate, weak, or non-biofilm producers based on OD570 values relative to negative controls.

Antibiotic susceptibility testing

The Kirby-Bauer disk diffusion method was performed on Mueller-Hinton agar (Oxoid CM0337) according to CLSI guidelines (Rai et al., 2023). Bacterial suspensions were adjusted to a 0.5 McFarland standard (~1×10⁸ CFU/mL). Antibiotic disks (Oxoid) included ciprofloxacin (5 μg), gentamicin (10 μg), colistin (10 μg), and doxycycline (30 μg). Plates were incubated at 37°C for 24 hours. Inhibition zones were measured in millimeters using digital calipers, with each test performed in triplicate. Results were interpreted as susceptible or resistant according to CLSI breakpoints.

Cytokine profiling

Whey fractions were obtained by centrifuging milk samples at 3000×g for 15 minutes, followed by filtration through 0.45 μm membranes. Concentrations of interleukin-6 (IL-6) and interleukin-10 (IL-10) were determined using commercial bovine-specific ELISA kits (Thermo Fisher Scientific; IL-6: ESS0029, IL-10: ESS0030), following the manufacturer’s protocols. Absorbance was read at 450 nm using a BioTek ELx808 microplate reader. Cytokine concentrations were calculated from standard curves, and all samples were assayed in triplicate.

Statistical analysis

Data were analyzed using GraphPad Prism version 9.0 (GraphPad Software, San Diego, CA, USA). Descriptive statistics (mean ± standard deviation) were used for continuous variables. Group comparisons for cytokine levels and antibiotic inhibition zones were performed using one-way ANOVA followed by Tukey’s post-hoc test. Categorical variables, such as prevalence and biofilm formation rates, were compared using the chi-square test. Statistical significance was set at p < 0.05 (Motulsky, 2011).

Results

Prevalence and identification of Staphylococcus isolates

Out of 40 raw milk samples analyzed, Staphylococcus aureus was identified in 25 samples (62.5%), while S. epidermidis was detected in 10 samples (25%). The remaining five samples (12.5%) were negative for staphylococcal growth. The difference in prevalence between the two species was statistically significant (χ² = 15.2, p < 0.001) (Table 1, Figure 1).

 

Table 1: Prevalence of Staphylococcus isolates in raw milk samples.

Species	Number of positive samples (%)	Total samples (n)
S. aureus	25 (62.5%)	40
S. epidermidis	10 (25.0%)	40
Negative	5 (12.5%)	40

 

Chi-square test showed a significant difference in prevalence between S. aureus and S. epidermidis (p < 0.001).

 

 

Biofilm formation capacity

Biofilm production was observed in 70% of S. aureus isolates (18/25) and 30% of S. epidermidis isolates (3/10), as determined by Congo Red Agar. The difference in biofilm-forming capacity between the two species was statistically significant (χ² = 8.3, p = 0.004) (Table 2, Figure 1).

 

Table 2: Biofilm formation by Staphylococcus isolates.

Species	Biofilm producers n (%)	Non-producers (%)	Total isolates
S. aureus	18 (72.0%)	7 (28.0%)	25
S. epidermidis	3 (30.0%)	7 (70.0%)	10

 

Chi-square test revealed a significantly higher proportion of biofilm producers among S. aureus compared to S. epidermidis (p = 0.004).

 

Antibiotic susceptibility patterns

Antibiotic susceptibility testing demonstrated marked differences in resistance profiles. S. aureus isolates exhibited a 42% resistance rate to ciprofloxacin, while resistance to gentamicin was only 5%. The mean inhibition zones for gentamicin (25.4 ± 1.2 mm) were significantly larger than those for ciprofloxacin (18.2 ± 0.9 mm), colistin (15.8 ± 1.1 mm), and doxycycline (20.6 ± 1.4 mm) (ANOVA F = 45.6, p < 0.001; Tukey’s post-hoc test) (Table 3, Figure 2).

 

Table 3: Antibiotic susceptibility of Staphylococcus isolates.

Antibiotic	Mean inhibition zone (mm) ± SD	Resistance rate (%)	CLSI Breakpoint (mm)	Interpretation
Gentamicin	25.4 ± 1.2	5	≤ 15	Susceptible
Ciprofloxacin	18.2 ± 0.9	42	≤ 21	High resistance
Colistin	15.8 ± 1.1	30	≤ 11	Moderate resistance
Doxycycline	20.6 ± 1.4	18	≤ 14	Moderate resistance

 

A one-way ANOVA revaled significant differences among antibiotics (p < 0.001). Tukey’s post-hoc test indicated gentamicin was significantly more effective than all other antibiotics (p < 0.05 for all pairwise comparisons).

 

Cytokine response profiles

Immunological analysis revealed that three concentrations of IL-6 and IL-10 were significantly elevated in milk samples inoculated with S. aureus compared to those with S. epidermidis and the control group. Mean IL-6 levels in the S. aureus group were 42.5 ± 3.2 pg/mL, compared to 28.9 ± 2.7 pg/mL for S. epidermidis and 10.2 ± 1.5 pg/mL for controls. Similarly, IL-10 levels were 15.3 ± 1.8 pg/mL for S. aureus, 10.4 ± 1.2 pg/mL for S. epidermidis, and 6.8 ± 0.9 pg/mL for controls. One-way ANOVA followed by Tukey’s post-hoc test confirmed these differences were highly significant (p < 0.001 for all comparisons) (Table 4).

 

Table 4: Cytokine concentrations in milk samples.

Group	IL-6 (pg/mL) Mean ± SD	IL-10 (pg/mL) Mean ± SD
S. aureus infected	42.5 ± 3.2	15.3 ± 1.8
S. epidermidis	28.9 ± 2.7	10.4 ± 1.2
Control	10.2 ± 1.5	6.8 ± 0.9

 

One-way ANOVA with Tukey’s post-hoc test showed significant elevations in both cytokines for S. aureus-infected samples compared to S. epidermidis and controls (p < 0.001).

 

 

Discussion

This study highlights significant microbial contamination in raw milk, with Staphylococcus aureus (62.5%) and S. epidermidis (25%) prevalence underscoring the contamination risks associated with hygiene lapses. Selective media (MSA and CRA) confirmed biofilm formation in 70% of S. aureus isolates, enhancing its persistence despite sanitation efforts (Yambise et al., 2020; Alam et al., 2020). The increased S. aureus prevalence surpasses European reports (De Vliegher et al., 2012), aligning instead with challenges in developing regions (Nickerson et al., 2009), such as limited diagnostics and inconsistent mastitis protocols in areas like Babylon/Iraq. While S. epidermidis signals environmental contamination, S. aureus poses greater risks due to production of enterotoxins (Marza et al., 2017; Abed et al., 2024). The observed antibiotic resistance patterns revealed a critical paradox, where the apparent paradox between inhibition zone sizes and resistance rates becomes clear upon examining CLSI breakpoints. The 42% resistance to ciprofloxacin, despite measurable inhibition zones (18.2 ± 0.9 mm), directly reflects the stringent breakpoint criteria (≤21 mm) for this critically important antimicrobial (Humphries et al., 2019), and mirrors the growing global concern about fluoroquinolone resistance in livestock-associated staphylococci (Hooper, 2021, Al-Isawi et al., 2020). In contrast, the maintained susceptibility to gentamicin (5% resistance) suggests that it may still serve as a viable treatment option in this region. However, though its use should be guided by regular susceptibility testing given the potential for aminoglycoside resistance gene transfer. The immunological findings demonstrate a robust host response to S. aureus infection, with IL-6 levels (42.5 ± 3.2 pg/mL) exceeding those in S. epidermidis infections by nearly 50%, supporting the concept of pathogen-specific immune activation patterns (Guan et al., 2020; Bochniarz., 2017; Chung et al., 2015). However, the simultaneous elevation of IL-10 (15.3 ± 1.8 pg/mL) suggests a concurrent anti-inflammatory response that may contribute to the establishment of chronic infection. This phenomenon warrants further investigation through longitudinal studies. These collective findings carry essential practical implications: The high prevalence of biofilm necessitates revised hygiene protocols that incorporate biofilm-disrupting agents, the resistance patterns require immediate antimicrobial stewardship programs focusing on fluoroquinolone restriction, and the cytokine profiles suggest potential for developing immune-based mastitis monitoring tools. While providing valuable insights, this study also highlights knowledge gaps that future research should address, particularly regarding the molecular mechanisms driving the observed high resistance rates and the potential for strain-specific variations in virulence factors across different dairy production systems.

Acknowledgements

We dedicate our scientific efforts to the Internal and Preventive Medicine, Microbiology and Veterinary Public Health Departments of the College of Veterinary Medicine at Al-Qasim Green University, Babylon, Iraq, for their support and ongoing encouragement, as well as for providing the research requirements that have facilitated to completion of this scientific research.

Novelty Statement

This study provides the first comprehensive assessment of Staphylococcus aureus and S. epidermidis prevalence, biofilm formation, antimicrobial resistance, and immune response profiles in raw milk from dairy farms in Babylon Governorate, Iraq. The findings highlight region-specific challenges, such as high ciprofloxacin resistance and robust cytokine responses, which are critical for tailoring interventions in similar resource-limited settings.

Author’s Contribution

Ali Dhiaa Marza conceptualized the study, developed the methodology, performed data analysis, and prepared the original draft of the manuscript. Amer Jebur Obayes AL-Isawi was responsible for sample collection, conducted laboratory analyses, and contributed to the review and editing of the manuscript. Noor R. Abady carried out the microbiological analyses and interpreted the resulting data. Ahmed Hassan Salh performed the immunological assays and handled statistical analysis. Faez Firdaus Jesse Abdullah supervised the research, secured funding, and finalized the manuscript. All authors reviewed and approved the final version of the manuscript. Financial Disclosure Statement.

Animal rights statement

Not applicable.

Ethical considerations

Sample collection was conducted as part of routine farm management procedures, without causing harm or requiring invasive intervention. Informed consent was obtained from all farm owners before participation.

Conflict of Interest

The authors have declared no conflict of interest in the publication of this manuscript.

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